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Biotech / Medical : Indications -- obesity/erectile dysfunction

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From: scaram(o)uche10/31/2019 11:48:11 AM
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Front Mol Neurosci. 2019 Sep 20;12:224. doi: 10.3389/fnmol.2019.00224. eCollection 2019.

Systematic Affinity Purification Coupled to Mass Spectrometry Identified p62 as Part of the Cannabinoid Receptor CB2 Interactome.

Sharaf A1, Mensching L1, Keller C1, Rading S1, Scheffold M1,2, Palkowitsch L3, Djogo N1, Rezgaoui M4, Kestler HA5, Moepps B2, Failla AV6, Karsak M1.

1
Neuronal and Cellular Signal Transduction, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
2
Institute of Pharmacology and Toxicology, Ulm University, Ulm, Germany.
3
Institute of Physiological Chemistry, Ulm University, Ulm, Germany.
4
Institute for Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany.
5
Institute of Medical Systems Biology, Ulm University, Ulm, Germany.
6
Microscopy Imaging Facility, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

The endocannabinoid system (ECS) consists particularly of cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, and enzymes that synthesize and degrade their ligands. It acts in a variety of organs and disease states ranging from cancer progression over neuropathic pain to neurodegeneration. Protein components engaged in the signaling, trafficking, and homeostasis machinery of the G-protein coupled CB2, are however largely unknown. It is therefore important to identify further interaction partners to better understand CB2 receptor functions in physiology and pathophysiology. For this purpose, we used an affinity purification and mass spectrometry-based proteomics approach of Strep-HA-CB2 receptor in HEK293 cells. After subtraction of background interactions and protein frequency library assessment we could identify 83 proteins that were classified by the identification of minimally 2 unique peptides as highly probable interactors. A functional protein association network analysis obtained an interaction network with a significant enrichment of proteins functionally involved in protein metabolic process, in endoplasmic reticulum, response to stress but also in lipid metabolism and membrane organization. The network especially contains proteins involved in biosynthesis and trafficking like calnexin, Sec61A, tubulin chains TUBA1C and TUBB2B, TMED2, and TMED10. Six proteins that were only expressed in stable CB2 expressing cells were DHC24, DHRS7, GGT7, HECD3, KIAA2013, and PLS1. To exemplify the validity of our approach, we chose a candidate having a relatively low number of edges in the network to increase the likelihood of a direct protein interaction with CB2 and focused on the scaffold/phagosomal protein p62/SQSTM1. Indeed, we independently confirmed the interaction by co-immunoprecipitation and immunocytochemical colocalization studies. 3D reconstruction of confocal images furthermore showed CB2 localization in close proximity to p62 positive vesicles at the cell membrane. In summary, we provide a comprehensive repository of the CB2 interactome in HEK293 cells identified by a systematic unbiased approach, which can be used in future experiments to decipher the signaling and trafficking complex of this cannabinoid receptor. Future studies will have to analyze the exact mechanism of the p62-CB2 interaction as well as its putative role in disease pathophysiology.

Molecules. 2019 Oct 12;24(20). pii: E3672. doi: 10.3390/molecules24203672.

Cannabinoid Receptor Interacting Protein 1a (CRIP1a): Function and Structure.

Booth WT1, Walker NB2, Lowther WT3,4, Howlett AC5,6.

1
Department of Biochemistry and Center for Structural Biology, Wake Forest School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA. wbooth@jcsu.edu.
2
Department of Physiology and Pharmacology, Wake Forest School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA. nbwalker@wakehealth.edu.
3
Department of Biochemistry and Center for Structural Biology, Wake Forest School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA. tlowther@wakehealth.edu.
4
Center for Molecular Signaling, Wake Forest University, 1834 Wake Forest Road, Winston-Salem, NC 27109, USA. tlowther@wakehealth.edu.
5
Center for Molecular Signaling, Wake Forest University, 1834 Wake Forest Road, Winston-Salem, NC 27109, USA. ahowlett@wakehealth.edu.
6
Department of Physiology and Pharmacology, Center for Research on Substance Use and Addiction, Wake Forest School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA. ahowlett@wakehealth.edu.

Cannabinoid receptor interacting protein 1a (CRIP1a) is an important CB1 cannabinoid receptor-associated protein, first identified from a yeast two-hybrid screen to modulate CB1-mediated N-type Ca2+ currents. In this paper we review studies of CRIP1a function and structure based upon in vitro experiments and computational chemistry, which elucidate the specific mechanisms for the interaction of CRIP1a with CB1 receptors. N18TG2 neuronal cells overexpressing or silencing CRIP1a highlighted the ability of CRIP1 to regulate cyclic adenosine 3',5'monophosphate (cAMP) production and extracellular signal-regulated kinase (ERK1/2) phosphorylation. These studies indicated that CRIP1a attenuates the G protein signaling cascade through modulating which Gi/o subtypes interact with the CB1 receptor. CRIP1a also attenuates CB1 receptor internalization via ß-arrestin, suggesting that CRIP1a competes for ß-arrestin binding to the CB1 receptor. Predictions of CRIP1a secondary structure suggest that residues 34-110 are minimally necessary for association with key amino acids within the distal C-terminus of the CB1 receptor, as well as the mGlu8a metabotropic glutamate receptor. These interactions are disrupted through phosphorylation of serines and threonines in these regions. Through investigations of the function and structure of CRIP1a, new pharmacotherapies based upon the CRIP-CB1 receptor interaction can be designed to treat diseases such as epilepsy, motor dysfunctions and schizophrenia.
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